The present invention, in some embodiments thereof, relates to molecular analysis and separation and, more particularly, but not exclusively, to methods and system for isoelectric focusing.
Isoelectric focusing is an analytical technique for separating molecules in an analyte sample by taking advantage of the differing ionic properties of the molecules.
Isoelectric focusing is usually performed in an electrolyte solution, optionally in a gel form, for example based on polyacrylamide, starch and/or agarose, having an immobilized proton concentration gradient, generally the proton concentration gradient changing from higher to lower pH in a given direction. In some implementation solutions which contain ampholytes which, under an electric field, generate a pH gradient. In isoelectric focusing, the separation takes place in a pH gradient that occupies the whole separation distance and is arranged so that the pH in the gradient increases from anode towards the cathode. In use, the analyte is loaded onto some location on the electrolyte solution. The charge of each different molecule changes in response to the ambient proton concentration according to the acidity (pKa) of the various functional groups of the molecule.
An electric potential is applied parallel to the proton concentration gradient between an isoelectric focusing anode and isoelectric focusing cathode. Molecules having a net positive charge migrate through the electrolyte solution towards the cathode while molecules having a net negative charge migrate through the electrolyte solution towards the anode.
As the molecules migrate, the ambient pH changes to reduce the net charge on the molecule until the molecule reaches an isoelectric point (pI) where, due to the ambient pH, the net charge on the molecule is zero. In this point the migrating molecule stops since they have zero charge. In such a manner, isoelectric focusing focuses molecules having a certain pI into a relatively narrow volume of the electrolyte solution. Isoelectric focusing is useful for the analysis of proteins by characterizing them according to their acidities. More importantly, it is useful for separation of protein mixtures.
International Patent Application Publication No. WO 2009/027970, published on Mar. 5, 2009 and incorporated herein by reference, describes methods and devices useful in producing local concentrations of protons, proton concentration gradients and desired proton concentration topographies in an environment, such as an electrolyte solution, a gel, or the like, including an electrolyte. This application also discloses methods and devices for isoelectric focusing and for display of data.